Interaction sites of the Epstein-Barr virus Zta transcription factor with the host genome in epithelial cells.

Epstein-Barr virus (EBV) is present in a state of latency in infected memory B-cells and EBV-associated lymphoid and epithelial cancers. Cell stimulation or differentiation of infected B-cells and epithelial cells induces reactivation to the lytic replication cycle. In each cell type, the EBV transcription and replication factor Zta (BZLF1, EB1) plays a role in mediating the lytic cycle of EBV. Zta is a transcription factor that interacts directly with Zta response elements (ZREs) within viral and cellular genomes. Here we undertake chromatin-precipitation coupled to DNA-sequencing (ChIP-Seq) of Zta-associated DNA from cancer-derived epithelial cells. The analysis identified over 14 000 Zta-binding sites in the cellular genome. We assessed the impact of lytic cycle reactivation on changes in gene expression for a panel of Zta-associated cellular genes. Finally, we compared the Zta-binding sites identified in this study with those previously identified in B-cells and reveal substantial conservation in genes associated with Zta-binding sites.

Identifying the Cellular Interactome of Epstein-Barr Virus Lytic Regulator Zta Reveals Cellular Targets Contributing to Viral Replication.

The human gammaherpesvirus Epstein-Barr virus (EBV) (human herpesvirus 4 [HHV4]) infects most adults and is an important contributor to the development of many types of lymphoid and epithelial cancers. Essential contributions of viral genes to viral replication are known, but the potential contributions of cell genes are less well delineated. A key player is the viral protein Zta (BZLF1, ZEBRA, or Z). This sequence-specific DNA-binding protein can disrupt EBV latency by driving the transcription of target genes and by interacting with the EBV lytic origin of replication. Here, we used an unbiased proteomics approach to identify the Zta-interactome in cells derived from Burkitt's lymphoma. Isolating Zta and associated proteins from Burkitt's lymphoma cells undergoing EBV replication, followed by tandem mass tag (TMT) mass spectrometry, resulted in the identification of 39 viral and cellular proteins within the Zta interactome. An association of Zta with the cellular protein NFATc2 was validated in independent experiments. Furthermore, the ability of Zta to attenuate the activity of an NFAT-dependent promoter was shown, which suggests a functional consequence for the association. The expression of Zta is itself regulated through NFAT activity, suggesting that Zta may contribute to a feedback loop that would limit its own expression, thus aiding viral replication by preventing the known toxic effects of Zta overexpression.IMPORTANCE Epstein-Barr virus infects most people across the world and causes several kinds of cancer. Zta is an important viral protein that makes the virus replicate by binding to its DNA and turning on the expression of some genes. We used a sensitive, unbiased approach to isolate and identify viral and cellular proteins that physically interact with Zta. This revealed 39 viral and cellular proteins. We found that one protein, termed NFATc2, was already known to be important for a very early step in viral replication. We identify that once this step has occurred, Zta reduces the effectiveness of NFATc2, and we suggest that this is important to prevent cells from dying before viral replication is complete and the mature virus is released from the cells.

Mechanism of activation of the BNLF2a immune evasion gene of Epstein-Barr virus by Zta.

The human gamma herpes virus Epstein-Barr virus (EBV) exploits multiple routes to evade the cellular immune response. During the EBV lytic replication cycle, viral proteins are expressed that provide excellent targets for recognition by cytotoxic T cells. This is countered by the viral BNLF2a gene. In B cells during latency, where BNLF2a is not expressed, we show that its regulatory region is embedded in repressive chromatin. The expression of BNLF2a mirrors the expression of a viral lytic cycle transcriptional regulator, Zta (BZLF1, EB1, ZEBRA), in B cells and we propose that Zta plays a role in up-regulating BNLF2a. In cells undergoing EBV lytic replication, we identified two distinct regions of interaction of Zta with the chromatin-associated BNLF2a promoter. We identify five potential Zta-response elements (ZREs) in the promoter that are highly conserved between virus isolates. Zta binds to these elements in vitro and activates the expression of the BNLF2a promoter in both epithelial and B cells. We also found redundancy amongst the ZREs. The EBV genome undergoes a biphasic DNA methylation cycle during its infection cycle. One of the ZREs contains an integral CpG motif. We show that this can be DNA methylated during EBV latency and that both Zta binding and promoter activation are enhanced by its methylation. In summary, we find that the BNLF2a promoter is directly targeted by Zta and that DNA methylation within the proximal ZRE aids activation. The implications for regulation of this key viral gene during the reactivation of EBV from latency are discussed.

Repression of CIITA by the Epstein-Barr virus transcription factor Zta is independent of its dimerization and DNA binding.

Repression of the cellular CIITA gene is part of the immune evasion strategy of the γherpes virus Epstein-Barr virus (EBV) during its lytic replication cycle in B-cells. In part, this is mediated through downregulation of MHC class II gene expression via the targeted repression of CIITA, the cellular master regulator of MHC class II gene expression. This repression is achieved through a reduction in CIITA promoter activity, initiated by the EBV transcription and replication factor, Zta (BZLF1, EB1, ZEBRA). Zta is the earliest gene expressed during the lytic replication cycle. Zta interacts with sequence-specific elements in promoters, enhancers and the replication origin (ZREs), and also modulates gene expression through interaction with cellular transcription factors and co-activators. Here, we explore the requirements for Zta-mediated repression of the CIITA promoter. We find that repression by Zta is specific for the CIITA promoter and can be achieved in the absence of other EBV genes. Surprisingly, we find that the dimerization region of Zta is not required to mediate repression. This contrasts with an obligate requirement of this region to correctly orientate the DNA contact regions of Zta to mediate activation of gene expression through ZREs. Additional support for the model that Zta represses the CIITA promoter without direct DNA binding comes from promoter mapping that shows that repression does not require the presence of a ZRE in the CIITA promoter.

Epstein-Barr virus transcription factor Zta acts through distal regulatory elements to directly control cellular gene expression.

Lytic replication of the human gamma herpes virus Epstein-Barr virus (EBV) is an essential prerequisite for the spread of the virus. Differential regulation of a limited number of cellular genes has been reported in B-cells during the viral lytic replication cycle. We asked whether a viral bZIP transcription factor, Zta (BZLF1, ZEBRA, EB1), drives some of these changes. Using genome-wide chromatin immunoprecipitation coupled to next-generation DNA sequencing (ChIP-seq) we established a map of Zta interactions across the human genome. Using sensitive transcriptome analyses we identified 2263 cellular genes whose expression is significantly changed during the EBV lytic replication cycle. Zta binds 278 of the regulated genes and the distribution of binding sites shows that Zta binds mostly to sites that are distal to transcription start sites. This differs from the prevailing view that Zta activates viral genes by binding exclusively at promoter elements. We show that a synthetic Zta binding element confers Zta regulation at a distance and that distal Zta binding sites from cellular genes can confer Zta-mediated regulation on a heterologous promoter. This leads us to propose that Zta directly reprograms the expression of cellular genes through distal elements.

Genome-wide analyses of Zta binding to the Epstein-Barr virus genome reveals interactions in both early and late lytic cycles and an epigenetic switch leading to an altered binding profile.

The Epstein-Barr virus (EBV) genome sustains substantial epigenetic modification involving chromatin remodelling and DNA methylation during lytic replication. Zta (ZEBRA, BZLF1), a key regulator of the EBV lytic cycle, is a transcription and replication factor, binding to Zta response elements (ZREs) in target promoters and EBV lytic origins of replication. In vitro, Zta binding is modulated by DNA methylation; a subset of CpG-containing Zta binding sites (CpG ZREs) is bound only in a DNA methylation-dependent manner. The question of how the dynamic epigenetic environment impacts Zta interaction during the EBV lytic cycle is unknown. To address this, we used chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) to identify Zta binding sites across the EBV genome before and after viral DNA replication. Replication did not alter the association of Zta across many regions of the EBV genome, but a striking reduction in Zta binding occurred at some loci that contain CpG ZREs. Separating Zta-bound DNA into methylated and nonmethylated fractions, we found that promoters that contain CpG ZREs were enriched in the methylated fraction but that Zta binding to promoters lacking CpG ZREs was not reduced. We hypothesize that the loss of DNA methylation on the EBV genome during the lytic cycle causes the reduced binding to CpG ZREs; this may act as a lytic cycle epigenetic switch. However, the epigenetic changes associated with the replicated EBV genome do not affect the interaction of Zta with many loci that are rich in non-CpG ZREs; this leads to sustained binding at these regions.

Dynamic chromatin environment of key lytic cycle regulatory regions of the Epstein-Barr virus genome.

The ability of Epstein-Barr virus (EBV) to establish latency allows it to evade the immune system and to persist for the lifetime of its host; one distinguishing characteristic is the lack of transcription of the majority of viral genes. Entry into the lytic cycle is coordinated by the viral transcription factor, Zta (BZLF1, ZEBRA, and EB1), and downstream effectors, while viral genome replication requires the concerted action of Zta and six other viral proteins at the origins of lytic replication. We explored the chromatin context at key EBV lytic cycle promoters (BZLF1, BRLF1, BMRF1, and BALF5) and the origins of lytic replication during latency and lytic replication. We show that a repressive heterochromatin-like environment (trimethylation of histone H3 at lysine 9 [H3K9me3] and lysine 27 [H3K27me3]), which blocks the interaction of some transcription factors with DNA, encompasses the key early lytic regulatory regions. Epigenetic silencing of the EBV genome is also imposed by DNA methylation during latency. The chromatin environment changes during the lytic cycle with activation of histones H3, H4, and H2AX occurring at both the origins of replication and at the key lytic regulatory elements. We propose that Zta is able to reverse the effects of latency-associated repressive chromatin at EBV early lytic promoters by interacting with Zta response elements within the H3K9me3-associated chromatin and demonstrate that these interactions occur in vivo. Since the interaction of Zta with DNA is not inhibited by DNA methylation, it is clear that Zta uses two routes to overcome epigenetic silencing of its genome.

A human 5'-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damage.

Topoisomerases regulate DNA topology and are fundamental to many aspects of chromosome metabolism. Their activity involves the transient cleavage of DNA, which, if it occurs near sites of endogenous DNA damage or in the presence of topoisomerase poisons, can result in abortive topoisomerase-induced DNA strand breaks. These breaks feature covalent linkage of the enzyme to the DNA termini by a 3'- or 5'-phosphotyrosyl bond and are implicated in hereditary human disease, chromosomal instability and cancer, and underlie the clinical efficacy of an important class of anti-tumour poisons. The importance of liberating DNA termini from trapped topoisomerase is illustrated by the progressive neurodegenerative disease observed in individuals containing a mutation in tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme that cleaves 3'-phosphotyrosyl bonds. However, a complementary human enzyme that cleaves 5'-phosphotyrosyl bonds has not been reported, despite the effect of DNA double-strand breaks containing such termini on chromosome instability and cancer. Here we identify such an enzyme in human cells and show that this activity efficiently restores 5'-phosphate termini at DNA double-strand breaks in preparation for DNA ligation. This enzyme, TTRAP, is a member of the Mg(2+)/Mn(2+)-dependent family of phosphodiesterases. Cellular depletion of TTRAP results in increased susceptibility and sensitivity to topoisomerase-II-induced DNA double-strand breaks. TTRAP is, to our knowledge, the first human 5'-tyrosyl DNA phosphodiesterase to be identified, and we suggest that this enzyme is denoted tyrosyl DNA phosphodiesterase-2 (TDP2).